Radiation therapy is widely used to treat cancer and kills cells by creating DNA double strand breaks (DSBs). DSB repair requires the loading of DNA repair proteins, including the ATM kinase, onto the chromatin at the site of damage. Chromatin is organized into distinct functional domains, including transcribed genes and silent heterochromatin, and the distinct chromatin architecture of these domains plays a key role in DSB repair. A critical modulator of chromatin function is the post-translational modification of histones by methylation. Many DNA-dependent events, including transcription and DNA replication, require the rapid rewriting of histone methylation signatures. Our current work demonstrates that ionizing radiation leads to the rapid methylation of histone H3 on lysine 9 (H3K9). This methylation of H3K9 occurs on large chromatin domains which extend at least 200kb on either side of the DSB, and are essential for maintaining activation of the Tip60 acetyltransferase and the ATM protein kinase. The central hypothesis is that DSBs created by ionizing radiation lead to the rapid rewriting of histone methylation codes at DSBs. Using Zinc Finger Nucleases to create sequence-specific DSBs in actively transcribed genes and inactive, intergenic regions, we will: (1) determine how H3K9 methylation signatures are rewritten in both actively transcribed genes and in intergenic regions of the chromatin~ (2) identify the methyltransferase which rewrites H3K9 methylation signatures and determine the mechanism by which this methyltransferase is loaded onto the chromatin at DSBs~ (3) determine how H3K9 methylation controls activation of Tip60 and ATM, and determine how loss of H3K9 methylation impacts the mechanism of DSB repair. By using Zinc Finger Nucleases and IR to create DSBs in defined chromatin domains, we can determine how DSB repair is influenced by both H3K9 methylation and by the local chromatin architecture at sites of damage. Further, since tumor cells exhibit alterations in chromatin organization and histone methylation signatures, this work will provide new insights into how changes in H3K9 methylation impact tumor sensitivity to radiation therapy. Understanding how the altered chromatin organization in tumor cells impacts radiosensitivity will therefore provide new insight into the intrinsic radioresistance of several tumor types to radiation therapy. PUBLIC HEALTH RELEVANCE: Radiation therapy is widely used to treat human cancer. By identifying the methyltransferase which methylate histones at DNA strand breaks, inhibitors of histone methyltransferases can be developed as novel radiosensitizers for use during radiation therapy. In addition, many tumors exhibit altered patterns of H3K9 methylation and deregulated expression of histone methyltransferases and demethylases. Understanding how altered patterns of H3K9 methylation in tumor cells influences DSB repair and intrinsic radiosensitivity of tumors can be used to guide appropriate cancer therapy choice for individual tumors based on their histone methylation profile.